B. pertussis infects the upper respiratory tract by adhering to ciliated epithelial cells and releasing toxins. The virulence genes that encode these proteins are regulated by the BvgS-BvgA two-component system. The response regulator BvgA binds to the promoter region of all known B. pertussis virulence genes to activate their transcription during infection. These genes include bipA, fha, ptx, prn, cya, bvgR, bvgA, and the fim genes. B. pertussis adhesin and adhesin-related genes, such as fim2, fim3, and fimX, are among the earliest genes activated by BvgA upon its induction. The Fim proteins facilitate adhesion of the bacterium to epithelial cells in the human respiratory tract. During transcription, the sigma subunit of RNA polymerase (RNAP) is the specificity factor that recognizes promoter elements. Primary sigmas like E. coli sigma70 and B. pertussis sigma A have specific regions that contact promoter sequences. Residues within Regions 4, 3, and 2 interact with the -35 element, the extended -10 sequence (positions -15, -14), and the -10 element, respectively. The fim3 promoter (Pfim3) has both the sigma70-dependent extended -10 sequence and a -10 element, while the fim2 promoter (Pfim2) has only a canonical -10 sequence. However, neither Pfim2 nor Pfim3 contains a recognizable -35 element. Instead each fim promoter contains a tract of cytosines (C), and the length of this C-tract regulates BvgA activation through phase-variation. The mechanism of BvgA-dependent regulation is not understood. We have collaborated with the lab of S. Stibitz (FDA) to examine in detail the DNA sequence elements involved in regulation of the promoters for fim2, fim3, and fimX. We have determined the optimum length of the C-tract for each promoter, and found that the three optimally configured promoters align perfectly, with identical distances between conserved upstream sequences and the downstream -10 element and the transcriptional start site. We have also demonstrated that the Pfim3 upstream conserved sequences correspond to BvgA-binding sites. The most upstream of two binding sites is predicted to be high affinity due to a nearly perfect match of one of its heptad half-sites to a functionally-derived consensus BvgA-binding sequence. The second binding site is a poor match to the consensus, with 10 of 14 bp belonging to the C-tract. Interestingly, this site of BvgA binding also corresponds exactly to the predicted position of the -35 element, typically recognized by sigma Region 4. However, the lack of a recognizable -35 element (CCCCCC vs. TTGACA), and the occupation of this site by BvgA suggest that activation of the fim3 promoter involves unusual interactions among BvgA, RNA polymerase, and promoter DNA.
